This project investigates novel interactions between the major secretory protein of catecholamine storage vesicles, chromogranin A (CgA), and the plasminogen/t-PA system, resulting in generation of active CgA peptide fragments which modulate (inhibit) catecholamine secretion. During the first 2.5 years of funding, we used a synthetic peptide approach, to identify a region within the primary sequence of CgA which has a pronounced effect on catecholamine secretion (here called catestatin ). Also, we demonstrated that components of the fibrinolytic system, tissue plasminogen activator (t-PA), is sorted into the soluble core of chromaffin vesicles, and is co-released along with CgA and catecholamines by exocytosis. In this proposal, we will test the hypothesis that the plasminogen/t-PA system is necessary and sufficient for generating CgA peptides specifically with catestatin sequence and activity. The specific aims are: Aim 1) to characterize the proteolytic effects of the plasminogen/t-PA system on CgA in vitro, identify the specific catestatin-containing peptides generated, and determine which of these peptides exhibit catestatin activity (inhibit catecholamine release from chromaffin cells). Our experimental approach will exploit recent advances in mass spectrometric techniques, including Matrix- assisted laser desorption/ionization mass spectrometry with time-of- flight analysis, a powerful and efficient method capable of determining specific peptide mass information even on samples containing complex mixtures of peptides. Aim 2) will determine the functional role of the endogenous plasminogen/t-PA system in generating catestatin peptide(s) by examining processing of CgA to peptide(s) with the catestatin sequence or portions of the catestatin sequence in the extracellular space of the chromaffin cell during secretion. Aim 3 will evaluate the effects of genetic manipulation of the plasminogen/t-PA system on generation of catestatin, and its effects on catecholamine release in vivo, using chromaffin cell lines which overexpress t-PA and transgenic mice deficient in plasminogen. In Aim 4 we will define the mechanism of action and functionally evaluate the peptides with catestatin sequence and activity generated by the action of plasmin on CgA, and test the hypothesis that the effects of the identified catestatin peptide(s) are specific for inhibition of nicotinic-mediated catecholamine release. In Aim 5 we will isolate the high affinity plasminogen receptor uniquely expressed by chromaffin cells and examine its role in the processing of CgA to catestatin containing peptides. These interactions between CgA and the plasminogen/t-PA system represent a heretofore unrecognized relationship between catecholaminergic and fibrinolytic pathways and a novel system which may have a dramatic impact upon catecholamine secretion and sympathoadrenal function.